The exact mechanism underlying chronic masseter muscle pain, a conspicuous symptom in temporomandibular disorder, remains unclear. We investigated whether expression of P2X3 receptor (P2X3R) is involved in mechanical hyperalgesia after contraction of masseter muscle (CMM). As compared with sham rats, the head-withdrawal threshold (HWT) to mechanical pressure stimulation of masseter muscle (MM) (but not after similar stimulation of facial skin) was significantly lower, and IL-1β level was significantly higher, in CMM rats on day 7 after CMM. The mean percentage of FG-labeled P2X3R-positive neurons was significantly increased in TG following successive IL-1β injections into the MM for 7 days. Successive administration of an IL-1β receptor-antagonist into the MM attenuated the increase of P2X3-IR cells in the TG. ATP release from MM after 300-g pressure stimulation of MM was also significantly enhanced after CMM. Administration into MM of the selective P2X3,2/3 receptor antagonist A-317491 attenuated the decrement of HWT in CMM rats. A significant increase in HWT was also observed at 30 min after A-317491 (60 µg) injection in IL-1β-injected rats. These findings suggest that P2X3R expression associated with enhanced IL-1β expression and ATP release in MM has a possible important role in MM mechanical hyperalgesia after excessive muscular contraction.